In 1972, Friedmann outlined the far-reaching opportunities for human gene therapy. Friedmann, T.; Roblin, R. Science 1972, 175, 949-955. Chromosomal deficiencies and/or anomalies, e.g., mutation and aberrant expression, cause many hereditary and non-hereditary diseases. Conventional medicine remains unable to treat many of these diseases; gene therapy may be an effective therapeutic option by either adding, replacing, or removing relevant genes. See Kay, M. A.; Liu, D.; Hoogergrugge, P. M. Proc. Natl. Acad. Sci. 1997, 94, 12744-12746 and Huang, L.; Hung, M.; Wagner, E., Eds. Nonviral Vectors for Gene Therapy; Academic Press: New York, 1999.
Currently few organs or cells can be specifically targeted for gene delivery. There are established protocols for transferring genes into cells, including calcium phosphate precipitation, electroporation, particle bombardment, liposomal delivery, viral-vector delivery, and receptor-mediated gene-delivery. However, a main obstacle to the penetration of a nucleic acid into a cell or target organ lies in its size and polyanionic nature, both of which militate against its passage across cell membranes. Two strategies currently being explored for delivery of nucleic acids are viral and synthetic non-viral vectors, i.e., cationic molecules and polymers. A brief discussion of viral vectors, cationic lipids, and cationic polymers and there utility in gene therapy is presented below.
Viral Vectors
Viral vectors are viruses. Viruses, such as adenoviruses, herpes viruses, retroviruses and adeno-associated viruses, are currently under investigation. Currently, viral vectors, e.g., adenoviruses and adeno-associated viruses, have exhibited the highest levels of transfection efficiency compared to synthetic vectors, i.e., cationic lipids and polymers. Viral vectors suffer use in the Treatment of Human Diseases Drugs 2000, 60, 249-271; Smith, E. A. Viral Vectors in Gene Therapy Annu. Rev. Microbiol. 1995, 49, 807-838; Drumm, M. L.; Pope, H. A.; Cliff, W. H.; Rommens, J. M.; Marvin, S. A.; Tsui, L. C.; Collins, F. S.; Frizzell, R. A.; Wilson, J. M. Correction of the Cystic-fibrosis Defect in Vitro by Retrovirus-Mediated Gene Transfer Cell 1990, 1990, 1227-1233; Rosenfeld, M. A.; Yoshimura, K.; Trapnell, B. C.; Yoneyama, K.; Rosenthal, E. R.; Dalemans, W.; Fukayama, M.; Bargon, J.; Stier, L. E.; Stratfordperricaudet, L.; Perricaudet, M.; Guggino, W. B.; Pavirani, A.; Lecocq, J. P.; Crystal, R. G. In vivo Transfer of the Human Cystic-Fibrosis Transmembrane Conductance Regulator Gene to the Airway Epithelium Cell 1992, 68, 143-155; Muzyczka, N. Use of Adenoassociated Virus as a General Transduction Vector for Mammalian Cells Curr. Top. Microbiol. Immuno. 1992, 158, 97-129; Robbins, P. D.; Tahara, H.; Ghivizzani, S. C. Viral Vectors for Gene Therapy Trends Biotechnol 1998, 16, 35-40; and oss, G.; Erickson, R.; Knorr, D.; Motulsky, A. G.; Parkman, R.; Samulski, J.; Straus, S. E.; Smith, B. R. Gene Therapy in the United States: A Five-Year Status Report Hum. Gene Ther. 1996, 14, 1781-1790.
Since the method infects an individual cell with a viral carrier, a potentially life threatening immune response to the treatment can develop. Summerford reviews gene therapy with Adeno-associated viral vectors. For additional details see Marshall, E. Clinical Research—FDA Halts All Gene Therapy Trials at Penn Science 2000, 287, 565-567 and Summerford, C.; Samulski, R. J. Adeno-associated Viral Vectors for Gene Therapy Biogenic Amines 1998, 14, 451-475. Several examples of viral vectors used for gene delivery are described below. In U.S. Pat. No. 5,585,362 to Wilson et al., an improved adenovirus vector and methods for making and using such vectors is described. Likewise, U.S. Pat. No. 6,268,213 to Samulski et al., describes an adeno-associated virus vector and cis-acting regulatory and promoter elements capable of expressing at least one gene and method of using the viral vector for gene therapy. Although the transfection efficiency is high with viral vectors, there are a number of complications associated with the use of viral vectors.
Cationic Lipids
The second strategy consists of using non-viral agents capable of promoting the transfer and expression of DNA in cells. Since the first report by Felgner, this area has been actively investigated. These cationic non-viral agents bind to polyanionic DNA. Following endocytosis, the nucleic acid must escape from the delivery agent as well as the endosomal compartment so that the genetic material is incorporated within the new host The mechanism of nucleic acid transfer from endosomes to cytoplasm and/or nuclear targets is still unclear. Possible mechanisms are simple diffusion, transient membrane destabilization, or simple leakage during a fusion event in which endosomes fuse with other vesicles. See Felgner, P. L. Nonviral Strategies for Gene Therapy Sci. Am. 1997, 276, 102-106; Felgner, P. L.; Gadek, T. R.; Holm, M.; Roman, R.; Chan, H. W.; Wenz, M.; Northrop, J. P.; Ringgold, G. M.; Danielsen, M. Lipofectin: A highly efficient, lipid mediated DNA-transfection procedure Proc. Natl. Acad. Sci. USA 1987, 84, 7413-7417; Felgner, P. L.; Kumar, R.; Basava, C.; Border, R. C.; Hwang-Felgner, J. In; Vical, Inc. San Diego, Calif.: U.S. Pat. No. 5,264,618, 1993; Felgner, J. H.; Kumar, R.; Sridhar, C. N.; Wheeler, C. J.; Tsai, Y. J.; Border, R.; Ramsey, P.; Martin, M.; Felgner, P. L. Enhanced Gene Delivery and Mechanism Studies with a Novel Series of Cationic Formulations J. Biol. Chem. 1994, 269, 2550-2561; Freidmann, T. Sci. Am. 1997, 276, 96-101; Behr, J. P. Gene Transfer with Synthetic Cationic Amphiphiles: Prospects for Gene Delivery Bioconjugate Chem. 1994, 5, 382-389; Cotton, M.; Wagner, B. Non-viral Approaches to Gene Therapy Curr. Op. Biotech. 1993, 4, 705-710; Miller, A. D. Cationic Liposomes for Gene Therapy Angew. Chem. Int. 1998, 37, 1768-1785; Scherman, D.; Bessodes, M.; Cameron, B.; Herscovici, J.; Hofland, H.; Pitard, B.; Soubrier, F.; Wils, P.; Crouzet, J. Application of Lipids and Plasmid Design for Gene Delivery to Mammalian Cells Curr. Op. Biotech. 1989, 9, 480; Lasic, D. D. In Surfactants in Cosmetics; 2nd ed.; Rieger, M. M., Rhein, L. D., Eds.; Marcel Dekker, Inc.: New York, 1997; Vol. 68, pp 263-283; Rolland, A. P. From Genes to Gene Medicines: Recent Advances in Nonviral Gene Delivery Crit. Rev. Ther. Drug 1998, 15, 143-198; de Lima, M. C. P.; Simoes, S.; Pires, P.; Faneca, H.; Duzgunes, N. Cationic Lipid-DNA Complexes in Gene Delivery from Biophysics to Biological Applications Adv. Drug. Del. Rev. 2001, 47, 277-294.
These synthetic vectors have two main functions, to condense the DNA to be transfected and to promote its cell-binding and passage across the plasma membrane, and where appropriate, the two nuclear membranes. Due to its polyanionic nature, DNA naturally has poor affinity for the plasma membrane of cells, which is also polyanionic. Several groups have reported the use of amphiphilic cationic lipid-nucleic acid complexes for in vivo transfection both in animals and humans. Thus, non-viral vectors have cationic or polycationic charges. See Gao, X; Huang, L. Cationic Liposome-mediated Gene Transfer Gene Therapy 1995, 2, 710-722; Zhu, N.; Liggott, D.; Liu, Y.; Debs, R. Systemic Gene Expression After Intravenous DNA Delivery into Adult Mice Science 1993, 261, 209-211; Thierry, A. R.; Lunardiiskandar, Y.; Bryant, J. L.; Rabinovich, P.; Gallo, R. C.; Mahan, L. C. Systemic Gene-Therapy-Biodistribution and Long-Term Expression of a Transgene in Mice Proc. Nat. Acad. Sci. 1995, 92, 9742-9746.
Cationic amphiphilic compounds that possess both cationic and hydrophobic domains have been previously used for delivery of genetic information. In fact, this class of compounds is widely used for intracellular delivery of genes. Such cationic compounds can form cationic liposomes which are the most popular system synthetic vector for gene transfection studies. The cationic liposomes serve two functions. First, it protects the DNA from degradation. Second, it increases the amount of DNA entering the cell. While the mechanisms describing how cationic liposomes function have not been fully delineated, such liposomes have proven useful in both in vitro and in vivo studies. Safinya, C. R. describes the structure of the cationic amphiphile-DNA complex. See Radler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810-814; Templeton, N. S.; Lasic, D. D.; Frederik, P. M.; Strey, H. H.; Roberts, D. D.; Pavlakis, G. N. Nature Biotech. 1997, 15, 647-652; Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. Science 1998, 281, 78-81; and Koltover, I.; Salditt, T.; Safinya, C. R. Biophys. J. 1999, 77, 915-924. Many of these systems for gene delivery in vitro and in vivo are reviewed in recent articles. See Remy, J.; Sirlin, C.; Vierling, P.; Behr, J. Bioconj. Chem. 1994, 5, 647-654; Crystal, R. G. Science 1995, 270, 404-410; Blaese, X.; et, a. Cancer Gene Ther. 1995, 2, 291-297; and Behr, J. P. and Gao, X cited above. Unlike viral vectors, the lipid-nucleic acid complexes can be used to transfer expression cassettes of essentially unlimited size.
Because these synthetic delivery systems lack proteins, they may evoke fewer immunogenic and inflammatory responses. However, the liposomes suffer from low transfection efficiencies. Moreover, as is the case with other polycations, cationic lipids and liposomes (e.g., Lipofectin®) can be toxic to the cells and inefficient in their DNA delivery in the presence of serum; see Leonetti et al. Behr, like Leonetti, reports that these cationic amphiphiles or lipids are adversely affected by serum and some are toxic. See Leonetti, J.; Machy, P.; Degols, G.; Lebleu, B.; Leserman, L. Proc. Nat. Acad. Sci. 1990, 87, 2448-2451 and Behr, J. P. Acc. Chem. Res. 1993, 26, 274-278.
Behr discloses numerous amphiphiles including dioctadecylamidologlycylspermine (“DOGS”) for gene delivery. This material is commercially available as TRANSFECTAM®. Vigneron describes guanidinium-cholesterol cationic lipids for transfection of eukaryotic cells. Felgner discloses use of positively-charged synthetic cationic lipids including N-1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (“DOTMA”), to form lipid/DNA complexes suitable for transfections. Byk describes cationic lipids where the cationic portion of the amphiphile is either linear, branched, or globular for gene transfection. Blessing and coworkers describe a cationic synthetic vector based on spermine. Safinya describes cationic lipids containing a poly(ethylene glycol) segment for gene delivery. Bessodes and coworkers describe a cationic lipid containing glycosidic linker for gene delivery. Ren and Liu describe cationic lipids based on 1,2,4-butanetriol. Tang and Scherman describe a cationic lipid that contains a disulfide linkage for gene delivery. Vierling describes highly fluorinated cationic amphiphiles as gene carrier and delivery systems. Jacopin describes a cation amphiphile for gene delivery that contains a targeting ligand. Wang and coworkers describe carnitine based cationic esters for gene delivery. Zhu describes the use of a cationic lipid, N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride for the intravenous delivery of DNA. See Behr, J. P.; Demeneix, B.; Loeffler, J. P.; Perez-Mutul, J. Efficeint Gene Transfer into Mammalian Primary Endocrine Cells with Lipopolyamine Coated DNA Proc. Nat. Acad. Sci. 1989, 86, 6982-6986; Vigneron, J. P.; Oudrhiri, N.; Fauquet, M.; Vergely, L.; Bradley, J. C.; Basseville, M.; Lehn, P.; Lehn, J. M. Proc. Nat. Acad. Sci. 1996, 93, 9682-9686; Byk, G.; BDubertret, C.; Escriou, V.; Frederic, M.; Jaslin, G.; Rangara, R.; Pitard, B.; Wils, P.; Schwartz, B.; Scherman, D. J. Med. Chem. 1998, 41, 224-235; Blessing, T.; Remy, J. S.; Behr, J. P. J. Am. Chem. Soc. 1998, 120, 8519-8520; Blessing, T.; Remy, J. S.; Behr, J. P. Proc. Nat. Acad. Sci. 1998, 95, 1427-1431; Schulze, U.; Schmidt, H.; Safinya, C. R. Bioconj. Chem. 1999, 10, 548-552; Bessodes, M.; Dubertret, C.; Jaslin, G.; Scherman, D. Bioorg. Med. Chem. Lett. 2000, 10, 1393-1395; Herscovici, J.; Egron, M. J.; Quenot, A.; Leclercq, F.; Leforestier, N.; Mignet, N.; Wetzer, B.; Scherman, D. Org. Lett. 2001; Ren, T.; Liu, D. Tetrahedron Lett. 1999, 40, 7621-7625; Tang, F.; Hughes, J. A. Biochem. Biophys. Res. Commun. 1998, 242, 141-145; Tang, F.; Hughes, J. A. Bioconjugate Chem. 1999, 10, 791-796; Wetzer, B.; Byk, G.; Frederic, M.; Airiau, M.; Blanche, F.; Pitard, B.; Scherman, D. Biochemical J. 2001, 356, 747-756; Vierling, P.; Santaella, C.; Greiner, J. J. Fluorine Chem. 2001, 107, 337-354; Jacopin, J.; Hofland, H.; Scherman, D.; Herscovici, J. J. Biomed. Chem. Lett. 2001, 11, 419-422; and Wang, J.; Guo, X.; Xu, Y.; Barron, L.; Szoka, F. C. J. Med. Chem. 1998, 41, 2207-2215.
In U.S. Pat. No. 5,283,185 to Epand et al., the inventors describe additional examples of amphiphiles including a cationic cholesterol synthetic vector, termed “DC-chol”. The inventors describe, in U.S. Pat. No. 5,264,6184, more cationic compounds that facilitate transport of biologically active molecules into cells. U.S. Pat. Nos. 6,169,078 and 6,153,434 to Hughes et al. disclose a cationic lipid that contains a disulfide bond for gene delivery. U.S. Pat. No. 5,334,761 to Gebeyehu et al. describes additional cationic amphiphiles suitable for intracellular delivery of biologically active molecules. U.S. Pat. No. 6,110,490 to Thierry describes additional cationic lipids for gene delivery. U.S. Pat. No. 6,056,938 to Unger, et al. discloses cationic lipid compounds that contain at least two cationic groups.
Cationic Polymers
Recently, polymeric systems for gene delivery have been explored. In Han's review, he discussed most of the common cationic polymer systems including PLL, poly(L-lysine); PEI, polyethyleneimine; pDMEAMA, poly(2-dimethylamino)ethyl-methacrylate; PLGA, poly(D,L-lactide-co-glycolide) and PVP (polyvinylpyrrolidone). See Garnett, M. C. Crit. Rev. Ther. Drug Carrier Sys. 1999, 16, 147-207; Han, S.; Mahato, R. I.; Sung, Y. K.; Kim, S. W. Molecular Therapy 2000, 2, 302-317; Zauner, W.; Ogris, M.; Wagner, E. Adv. Drug. Del. Rev. 1998, 30, 97-113; Kabanov, A. V.; Kabanov, V. A. Bioconj. Chem. 1995, 6, 7-20; Lynn, D. M.; Anderson, D. G.; Putman, D.; Langer, R. J. Am. Chem. Soc. 2001, 123, 8155-8156; Boussif, O.; Lezoualc'h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. USA 1995, 92, 7297-7301; Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480; Putnam, D.; Langer, R. Macromolecules 1999, 32, 3658-3662; Gonzalez, M. F.; Ruseckaite, R. A.; Cuadrado, T. R. Journal of Applied Polymer Science 1999, 71, 1223-1230; Tang, M. X.; Redemann, C. T.; Szoka, F. C. In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers Bioconjugate Chem. 1996, 7, 703-714; Kukowska-latallo, J. F.; Bielinska, A. U.; Johnson, J.; Spinder, R.; Tomalia, D. A.; Baker, J. R. Proc. Nat. Acad. Sci. 1996, 93, 4897-4902; and Lim, Y.; Kim, S.; Lee, Y.; Lee, W.; Yang, T.; Lee, M.; Suh, M.; Park, J. J. Am. Chem. Soc. 2001, 123, 2460-2461.
Some representative examples of cationic polymers under investigation are described below. For example, poly(β-amino esters) have been explored and shown to condense plasmid DNA into soluble DNA/polymer particles for gene delivery. To accelerate the discovery of synthetic transfection vectors parallel synthesis and screening of a cationic polymer library was reported by Langer. Wolfert describes cationic vectors for gene therapy formed by self-assembly of DNA with synthetic block cationic co-polymers. Haensler and Szoka describe the use of cationic dendrimer polymers (polyamidoamine (PAMAM) dendrimers) for gene delivery. Wang describes a cationic polyphosphoester for gene delivery. Putnam describes a cationic polymer containing imidazole for the delivery of DNA. See Lynn, D. M.; Langer, R. J. Am. Chem. Soc. 2000, 122, 10761-10768; Wolfert, M. A.; Schacht, E. H.; Toncheva, V.; Ulbrich, K.; Nazarova, O.; Seymour, L. W. Hum. Gene Ther. 1996, 7, 2123-2133; Haensler, J.; Szoka, F. Bioconj. Chem. 1993, 4, 372; and Wang, J.; Mao, H. Q.; Leong, K W. J. Am. Chem. Soc. 2001; Putnam, D.; Gentry, C. A.; Pack, D. W.; Langer, R. Proc. Nat. Acad. Sci. 2001, 98, 1200-1205.
A number of patents are also known that describe cationic polymers for gene delivery. For example, U.S. Pat. No. 5,629,184 to Goldenberg et al. describes cationic copolymers of vinylamine and vinyl alcohol for the delivery of oligonucleotides. U.S. Pat. No. 5,714,166 to Tomalia, et al, discloses dendritic cationic-amine-terminated polymers for gene delivery. U.S. Pat. No. 5,919,442 to Yin et al. describes cationic hyper comb-branched polymer conjugates for gene delivery. U.S. Pat. No. 5,948,878 to Burgess et al. describes additional cationic polymers for nucleic acid transfection and bioactive agent delivery. U.S. Pat. No. 6,177,274 to Park et al. discloses a compound for targeted gene delivery that consists of polyethylene glycol (PEG) grafted poly(L-lysine) (PLL) and a targeting moiety, wherein at least one free amino function of the PLL is substituted with the targeting moiety, and the grafted PLL contains at least 50% unsubstituted free amino function groups. U.S. Pat. No. 6,210,717 to Choi et al. describes a biodegradable, mixed polymeric micelle used to deliver a selected nucleic acid into a targeted host cell that contains an amphiphilic polyester-polycation copolymer and an amphiphilic polyester-sugar copolymer. U.S. Pat. No. 6,267,987 to Park et al. discloses a positively charged poly[alpha-(omega-aminoalkyl) glycolic acid] for the delivery of a bioactive agent via tissue and cellular uptake. U.S. Pat. No. 6,200,956 to Scherman et al. describes a pharmaceutical composition useful for transfecting a nucleic acid containing a cationic polypeptide.
All of these polymers possess and rely on cationic moieties to bind DNA. Thus, the need exits for non-cationic polymers or macromolecules for gene delivery. Such polymers would also be advantageous over using viral vectors because the polymer delivery system would not expose the cell to a virus that could infect the cell.
The following is only a representative description of the potential therapeutic value of gene therapy. Gene therapy can be used for cancer treatment with recent papers describing its utility for prostate, colorectal, ovarian, lung, breast cancer. Gene therapy has been explored for delivery of vaccines for infectious disease, for lysosomal storage disorders, for dendritic cell-based immunotherapy, for controlling hypertension, and for rescuing ischaemic tissues. Gene therapy has also been explored for treating HIV. See Galanis, E.; Vile, R.; Russell, S. J. Crit. Rev. Oncol. Hemat 2001, 38, 177-192; Kim, D.; Martuza, R. L.; Zwiebel, J. Nature Med. 2001, 7, 783-789; Culver, K W.; Blaese, R. M. Trends Genet 1994, 10, 174-178; Harrington, K J.; Spitzweg, C.; Bateman, A. R.; Morris, J. C.; Vile, R. G. J. Urology 2001, 166, 1220-1233; Chen, M. J.; Chung-Faye, G. A.; Searle, P. F.; Young, L. S.; Kerr, D. J. Biodrugs 2001, 15, 357-367; Wen, S. F.; Mahavni, V.; Quijano, E.; Shinoda, J.; Grace, M.; Musco-Hobkinson, M. L.; Yang, T. Y.; Chen, Y. T.; Runnenbaum, I.; Horowitz, J.; Maneval, D.; Hutchins, B.; Buller, R. Cancer Gene Ther. 2003, 10, 224-238; Hoang, T.; Traynor, A. M.; Schiller, J. H. Surg. Oncol. 2002, 11, 229-241; Patterson, A.; Harris, A. L. Drugs Aging 1999, 14, 75-90; Clark, K. R.; Johnson, P. R. Curr. Op. Mol. Ther. 2001, 3, 375-384; Yew, N. S.; Cheng, S. H. Curr. Op. Mol. Ther. 2001, 3, 399-406; Jenne, L.; Schuler, G.; Steinkasserer, A. Trends Immunol 2001, 22; Sellers, K. W.; Katovich, M. J.; Gelband, C. H.; Raizada, M. K. Am. J. Med. Sci. 2001, 322, 1-6; Emanueli, C.; Madeddu, P. Brit. J. Pharmacol. 2001, 133, 951-958; and Schnell, M. J. FEMS Microbiol Lett 2001, 200,123-129.
Therefore, the need exists for new compositions and methods for gene delivery. New gene delivery compositions will find applications in medicine and gene research. The present invention fulfills this need and has other related advantages.